PROJECT ABSTRACT One of the hallmark features of cancer is the alteration of cellular glycosylation. The transition of localized cancer to metastatic disease, which commonly leads to poor prognosis, critically features patent changes in glycosylation. Metastatic cancer cells synthesize aberrant levels and variable structures of glycans compared with their non-metastatic counterparts. Metastasis-associated glycans can modify cellular activities, such as adhesion, invasion and proliferation, as well as encourage binding of pro-metastatic lectins. Hence, metastasis-associated glycans and their related glycan-synthesizing enzymes offer attractive targets for therapeutic interference of cancer progression. In accordance with this collaborative UO1 tumor glycomics directive, we seek to leverage our complementary expertise and laboratory advances on analytics of cell surface glycans and on tumor glycobiology to examine how modifications in glycan structure influence malignant progression. Our guiding hypothesis is that acquisition of distinct metastasis-associated glycans impacts malignant behavior and metastatic potential. In preliminary studies, gene set enrichment analysis performed across (9) common human cancer types indicated that metastatic melanomas, above all, were enriched for a `glycome' gene signature. Subsequent glycomic analysis indicated that metastatic melanoma cells uniquely displayed a high level of i-linear poly-N-acetyllactosamines (polylacs), whereas normal human melanocytes almost exclusively expressed I-branched polylacs. Further comparative glycome gene profiling between normal melanocytes and metastatic melanoma cells identified suppressed levels of I-branching 1,6 N-acetylglucosaminyltransferase (GCNT2) in metastatic melanoma cells that was significantly correlated with melanoma progression. Enforced GCNT2 expression in human melanoma models strongly indicated that I- branch/GCNT2 caused significant attenuation of melanoma growth in vivo and of cell signaling and proliferation associated with insulin-like growth factor-1 receptor and 1 integrins. I-branching GCNT2 activity also inhibited binding of pro-melanoma metastasis lectin, galectin (Gal)-3. In this grant, we will leverage these exciting melanoma glycomic data to investigate the role of i-linear/I-branch polylacs and GCNT2 as regulators of metastatic melanoma behavior. The Specific Aims are: (1) To analyze function of I-branched glycans and GCNT2 on melanoma cell activity and (2) To determine whether I-branch/GCNT2 expression predicts melanoma progression. These Aims will be explored by a collaborative team of experts in tumor glycobiology, glyco-analytics and melanoma pathology and include the use of clinically-relevant human and mouse melanoma models and innovative histo-pathological, glyco-analytical and gene regulation systems. Our far-reaching goal is to understand how melanomas metastasize - the causal step of melanoma lethality in patients. Importantly, our findings will offer new insights into how glycomic regulators can be targeted for therapeutic exploitation or used as biomarkers to predict melanoma metastasis and clinical outcome in patients.